The goal of this translational project is to optimize targeted T-cells with chimeric antigen receptors (CARs) for use in mesothelioma. CARs are now beginning to show activity in a number of pilot clinical trials, however two issues have emerged that provide a barrier to rapid progress in the field: 1) available preclinical models have not accurately predicted the safety of CARs, and unexpected toxicities from cytokine release and tissue damage has been reported in recent trials; 2) high costs and long lead times required for vector production have slowed the clinical application of T cells expressing CARs, and prevent a facile and iterative approach to optimize CAR design and determine the optimal target structures on tumor cells. Our preliminary data establishes that T lymphocytes can be efficiently modified by mRNA electroporation without integration-associated safety concerns, and that repeated infusions of RNA CAR T cells mediate robust antitumor effects in preclinical humanized models with disseminated tumor xenografts. Thus, this new platform affords the possibility of rapidly testing potent RNA CARs for antitumor activity, and in the event of toxicity, limiting off-target exposure by discontinuing CAR administration. Because late relapses due to tumor escape variants in pre-clinical models have been identified, it will be important to test combinations of CAR T cells to augment antitumor effects and prevent recurrence. The specific aims are to 1) Develop a new platform technology using RNA engineering to create RNA CARs, 2) Conduct experiments using patient material from Project 1 to assess the effects of third generation CARs, and 3) Test the anti-tumor activity of a combinatorial CAR-based immunotherapy using CARs specific for mesothelin, high molecular weight melanoma-associated (HMW-MAA) and c-Met for mesothelioma. These studies will test the central hypothesis that multiple CARs (i.e., a CAR fleet) will improve CAR immunotherapy compared to therapy with monoclonal CARs. Furthermore, these studies will establish the safety and feasibility of increasing the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors.